(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`(43) International Publication Date
`12 December 2002 (12.12.2002)
`
`PCT
`
`(10) International Publication Number
`WO 02/098416 A2
`
`A61K 31/395,
`(51) International Patent Classification7:
`311675, A61P 35100 II (A61K 311395, 31:675)
`
`(21) International Application Number: PCT/US02/16737
`
`(22) International Filing Date:
`
`29 May 2002 (29.05.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`601295,236
`601295,190
`
`English
`
`English
`
`1 June 2001 (01.06.2001) US
`1 June 2001 (01.06.2001) US
`
`(71) Applicant: WYETH [US/US]; Five Giralda Farms, Madi(cid:173)
`son, NJ 07940 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, UZ, VN,
`YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR,
`NE, SN, TD, TG).
`
`(72) Inventors: DUKART, Gary; 1714 Benjamin Drive, Am(cid:173)
`bler, PA 19002 (US). GIBBONS, James, Joseph, Jr.; 33
`Terrace Drive, Westwood, NJ 07675 (US).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`(74) Agents: MILOWSKY, Arnold, S.; Wyeth, Patent Law
`Department, Five Giralda Farms, Madison, NJ 07940-0874
`et al. (US).
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`-iiiiiiii
`iiiiiiii -
`---
`!!!!!!! -
`!!!!!!!! -iiiiiiii
`iiiiiiii ----
`
`-!
`
`iiiiiiii
`
`\0
`,....i
`~
`QO
`
`...........
`
`~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`
`°" Q
`0 > (57) Abstract: This invention provides the use of a combination of an mTOR inhibitor and an antinoeplastic alkylating agent in the
`
`0
`
`(54) Title: ANTINEOPLASTIC COMBINATIONS
`
`~ treatment of neoplasms.
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`ANTINEOPLASTIC COMBINATIONS
`
`This invention relates to the use of combinations of an mTOR inhibitor (e.g
`rapamycin 42-ester with
`3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
`(CCl-779)) and an antineoplastic alkylating agent in the treatment of neoplasms, to
`the use of an mTOR inhibitor and an antineoplastic alkylating agent in the preparation
`of a medicament for the treatment of a neoplasm, to a product comprising an mTOR
`inhibitor and an antineoplastic alkylating agent as a combined preparation for
`simultaneous, separate or sequential use in the treatment of a neoplasm, and to
`pharmaceutical compositions comprising an mTOR
`inhibitor, an antineoplastic
`alkylating agent and a pharmaceutically acceptable carrier.
`
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`BACKGROUND OF THE INVENTION
`Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces
`hygroscopicus, which was found to have antifungal activity, particularly against
`Candida albicans, both in vitro and in vivo [C. Vezina et al., J. Antibiot. 28, 721 (1975);
`S.N. Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, 539
`(1978); U.S. Patent 3,929,992; and U.S. Patent 3,993,749]. Additionally, rapamycin
`alone (U.S. Patent 4,885, 171) or in combination with picibanil (U.S. Patent 4,401,653)
`has been shown to have antitumor activity.
`The immunosuppressive effects of rapamycin have been disclosed in FASEB
`3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules, also have
`been shown to be effective as immunosuppressive agents, therefore useful in
`preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y.
`25 Caine et al., Lancet 1183 (1978); and U.S. Patent 5, 100,899]. R. Martel et al. [Can. J.
`Physiol. Pharmacol. 55, 48 (1977)] disclosed that rapamycin is effective in the
`experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the
`adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the
`formation of lgE-like antibodies.
`lupus
`treating systemic
`in preventing or
`Rapamycin
`is also useful
`erythematosus
`[U.S. Patent 5,078,999], pulmonary
`inflammation
`[U.S. Patent
`5,080,899],
`insulin dependent diabetes mellitus
`[U.S. Patent 5,321,009], skin
`disorders, such as psoriasis [U.S. Patent 5,286,730], bowel disorders [U.S. Patent
`5,286,731], smooth muscle cell proliferation and intimal thickening following vascular
`
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`injury [U.S. Patents 5,288,711 and 5,516,781], adult T-cell leukemia/lymphoma
`inflammation
`[U.S. Patent
`[European Patent Application 525,960 A1], ocular
`5,387,589], malignant carcinomas [U.S. Patent 5,206,018], cardiac inflammatory
`disease [U.S. Patent 5,496,832], and anemia [U.S. Patent 5,561, 138].
`Rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
`(CCl-779) is ester of rapamycin which has demonstrated significant inhibitory effects
`on tumor growth in both in vitro and in vivo models. The preparation and use of
`in U.S. Patent
`hydroxyesters of rapamycin,
`including CCl-779, are disclosed
`5,362,718.
`CCl-779 exhibits cytostatic, as opposed to cytotoxic properties, and may delay
`the time to progression of tumors or time to tumor recurrence. CCl-779 is considered
`to have a mechanism of action that is similar to that of sirolimus. CCl-779 binds to
`and forms a complex with the cytoplasmic protein FKBP, which inhibits an enzyme,
`mTOR (mammalian
`target of rapamycin, also known as FKBP12-rapamycin
`associated protein [FRAP]).
`Inhibition of mTOR's kinase activity inhibits a variety of
`signal
`transduction pathways,
`including cytokine-stimulated cell proliferation,
`translation of mRNAs for several key proteins that regulate the G1 phase of the cell
`cycle, and IL-2-induced transcription, leading to inhibition of progression of the cell
`
`cycle from m to S. The mechanism of action of CCl-779 that results in the GI S
`
`phase block is novel for an anticancer drug.
`In vitro, CCl-779 has been shown to inhibit the growth of a number of
`histologically diverse tumor cells. Central nervous system (CNS) cancer, leukemia
`(T-cell), breast cancer, prostate cancer, and melanoma lines were among the most
`sensitive to CCl-779. The compound arrested cells in the G1 phase of the cell cycle.
`In vivo studies in nude mice have demonstrated that CCl-779 has activity
`against human tumor xenografts of diverse histological types. Gliomas were
`particularly sensitive to CCl-779 and the compound was active in an orthotopic glioma
`model in nude mice. Growth factor (platelet-derived)-induced stimulation of a human
`glioblastoma cell line in vitro was markedly suppressed by CCl-779. The growth of
`several human pancreatic tumors in nude mice as well as one of two breast cancer
`lines studied in vivo also was inhibited by CCl-779.
`
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`DESCRIPTION OF THE INVENTION
`This invention provides the use of combinations of an mTOR inhibitor and an
`antineoplastic alkylating agent as antineoplastic combination chemotherapy.
`In
`particular, these combinations are useful in the treatment of renal cancer, soft tissue
`cancer, breast cancer, neuroendocrine tumor of the lung, cervical cancer, uterine
`cancer, head and neck cancer, glioma, non-small lung cell cancer, prostate cancer,
`pancreatic cancer, lymphoma, melanoma, small cell lung cancer, ovarian cancer,
`colon cancer, esophageal cancer, gastric cancer, leukemia, colorectal cancer, and
`unknown primary cancer. This invention also provides combinations of an mTOR
`inhibitor and an antineoplastic alkylating agent for use as antineoplastic combination
`chemotherapy, in which the dosage of either the mTOR inhibitor or the antineoplastic
`alkylating agent or both are used in subtherapeutically effective dosages.
`
`In another aspect, the invention provides the use of combinations of an mTOR
`inhibitor and an antineoplastic alkylating agent in the preparation of a medicament for
`In a further aspect, the invention provides a product
`the treatment of a neoplasm.
`comprising an mTOR inhibitor and an antineoplastic alkylating agent as a combined
`preparation for simultaneous, separate or sequential use in the treatment of a
`neoplasm in a mammal.
`In a still further aspect, the invention provides a
`pharmaceutical composition comprising an mTOR
`inhibitor, an antineoplastic
`alkylating agent and a pharmaceutically acceptable carrier.
`
`As used in accordance with this invention, the term "treatment" means treating
`a mammal having a neoplastic disease by providing said mammal an effective amount
`of a combination of an mTOR inhibitor and an antineoplastic alkylating agent with the
`purpose of inhibiting growth of the neoplasm in such mammal, eradication of the
`neoplasm, or palliation of the mammal.
`
`As used in accordance with this invention, the term "providing," with respect to
`providing the combination, means either directly administering the combination, or
`administering a prodrug, derivative, or analog of one or both of the components of the
`combination which will form an effective amount of the combination within the body.
`
`mTOR is the mammalian target of rapamycin, also known as FKBP12-
`rapamycin associated protein [FRAP].
`Inhibition of mTOR's kinase activity inhibits a
`variety of signal transduction pathways, including cytokine-stimulated cell proliferation,
`
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`translation of mRNAs for several key proteins that regulate the G1 phase of the cell
`cycle, and IL-2-induced transcription, leading to inhibition of progression of the cell
`cycle from G1 to S.
`mTOR regulates the activity of at least two proteins involved in the translation
`of specific cell cycle regulatory proteins (Burnett, P.E., PNAS 95: 1432 (1998) and
`lsotani, S., J. Biol. Chem. 274: 33493 (1999)). One of these proteins p70s6 kinase is
`phosphorylated by mTOR on serine 389 as well as
`threonine 412.
`This
`phosphorylation can be observed in growth factor treated cells by Western blotting of
`whole cell extracts of these cells with antibody specific for the phosphoserine 389
`residue.
`As used in accordance with this invention, an "mTOR inhibitor" means a
`compound or ligand which inhibits cell replication by blocking progression of the cell
`cycle from G1 to S by inhibiting the phosphorylation of serine 389 of p70s6 kinase by
`mTOR.
`
`The following standard pharmacological test procedure can be used to
`determine whether a compound is an mTOR inhibitor, as defined herein. Treatment of
`growth factor stimulated cells with an mTOR inhibitor like rapamycin completely
`blocks phosphorylation of serine 389 as evidenced by Western blot and as such
`constitutes a good assay for mTOR inhibition. Thus whole cell lysates from cells
`stimulated by a growth factor (eg. IGF1) in culture in the presence of an mTOR
`inhibitor should fail to show a band on an acrylamide gel capable of being labeled with
`an antibody specific for serine 389 of p70s6K.
`
`5
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`25 Materials:
`NuPAGE LDS Sample Buffer
`NuPAGE Sample Reducing Agent
`NuPAGE 4-12% Bis-Tris Gel
`NuPAGE MOPS SOS Running Buffer
`30 Nitrocellulose
`NuPAGE Transfer Buffer
`Hyperfilm ECL
`ECL Western Blotting Detection Reagent
`
`(Novex Cat# NP0007)
`(Novex Cat# NP0004)
`(Novex Cat# NP0321)
`(Novex Cat# NP0001)
`(Novex Cat # LC2001)
`(Novex Cat # NP0006)
`(Amersham Cat# RPN3114H)
`(Amersham Cat# RPN2134)
`
`35
`
`(Cell Signaling Cat #9205)
`Phospho-p70 S6 Kinase (Thr389)
`Primary antibody:
`Secondary antibody: Goat anti-rabbit lgG-HRP conjugate (Santa Cruz Cat #sc-2004)
`
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`5
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`25
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`30
`
`Methods:
`A. Preparation of Cell Lysates
`Cell lines were grown in optimal basal medium supplemented with 10% fetal
`bovine serum and penicillin/treptomycin. For phosphorylation studies, cells were
`subcultured in 6-well plates. After the cells have completely attached, they were
`either serum-starved.
`Treatment with mTOR inhibitors ranged from 2 to 16
`hours. After drug treatment, the cells were rinsed once with PBS (phosphate
`
`buffered saline without Mg++ and Ca++) and then lysed in 150-200 µI NuPAGE
`
`sonicated and then
`LOS sample buffer per well. The lysates were briefly
`centrifuged for 15 minutes at 14000 rpm. Lysates were stored at minus -so0c until
`use.
`The test procedure can also be run by incubating the cells in growth medium
`overnight after they have completely attached. The results under both sets of
`conditions should be the same for an mTOR inhibitor.
`
`B. Western Blot Analysis
`
`1) Prepare total protein samples by placing 22.5 µI of lysate per tube and then
`
`add 2.5 µI NuPAGE sample reducing agent. Heat samples at 10°c for 10
`
`minutes. Electrophoresed using NuPAGE gels and NuPAGE SOS buffers.
`2) Transfer the gel to a nitrocellulose membrane with NuPAGE transfer buffer.
`The membrane are blocked for 1 hour with blocking buffer (Tris buffered
`saline with 0.1 %-Tween and 5% nonfat-milk). Rinse membranes 2x with
`washing buffer (Tris buffered saline with 0.1%-Tween).
`'
`3) Blots/membrane are incubated with the P-p70 S6K (T389) primary antibody
`
`(1:1000) in blocking buffer overnight at 4 C in a rotating platform.
`
`0
`
`4) Blots are rinsed 3x for 10 minutes each with washing buffer, and incubated
`with secondary antibody (1 :2000) in blocking buffer for 1 hour at room
`temperature.
`5) After the secondary antibody binding, blots are washed 3x for 10 minutes
`each with washing buffer, and 2x for 1 minute each with Tris-buffered saline,
`followed by chemiluminescent
`(ECL) detection and
`then exposed
`to
`chemiluminescence films.
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`As used in accordance with this invention, the term "a rapamycin" defines a
`class of immunosuppressive compounds which contain the basic rapamycin nucleus
`(shown below). The rapamycins of this invention include compounds which may be
`chemically or biologically modified as derivatives of the rapamycin nucleus, while still
`the term "a rapamycin"
`retaining
`immunosuppressive properties. Accordingly,
`includes esters, ethers, oximes, hydrazones, and hydroxylamines of rapamycin, as
`well as rapamycins in which functional groups on the rapamycin nucleus have been
`modified, for example through reduction or oxidation. The term "a rapamycin" also
`includes pharmaceutically acceptable salts of rapamycins, which are capable of
`forming such salts, either by virtue of containing an acidic or basic moiety.
`
`5
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`
`0
`
`OMe
`I
`
`MeO"
`
`RAPAMYCIN
`
`15
`
`20
`
`It is preferred that the esters and ethers of rapamycin are of the hydroxyl
`groups at the 42- and/or 31-positions of the rapamycin nucleus, esters and ethers of a
`hydroxyl group at the 27-position (following chemical reduction of the 27-ketone), and
`that the oximes, hydrazones, and hydroxylamines are of a ketone at the 42-position
`(following oxidation of the 42-hydroxyl group) and of 27-ketone of the rapamycin
`nucleus.
`
`Preferred 42- and/or 31-esters and ethers of rapamycin are disclosed in the
`following patents, which are all hereby incorporated by reference: alkyl esters (U.S.
`
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`Patent 4,316,885); aminoalkyl esters (U.S. Patent 4,650,803); fluorinated esters (U.S.
`Patent 5, 100,883); amide esters (U.S. Patent 5, 118,677); carbamate esters (U.S.
`Patent 5, 118,678); silyl ethers (U.S. Patent 5, 120,842); aminoesters (U.S. Patent
`5, 130,307); acetals (U.S. Patent 5,51,413); aminodiesters (U.S. Patent 5, 162,333);
`sulfonate and sulfate esters (U.S. Patent 5, 177,203); esters (U.S. Patent 5,221,670);
`alkoxyesters (U.S. Patent 5,233,036); 0-aryl, -alkyl, -alkenyl, and -alkynyl ethers (U.S.
`Patent 5,258,389); carbonate esters (U.S. Patent 5,260,300); arylcarbonyl and
`alkoxycarbonyl carbamates (U.S. Patent 5,262,423); carbamates (U.S. Patent
`5,302,584); hydroxyesters (U.S. Patent 5,362,718); hindered esters (U.S. Patent
`5,385,908); heterocyclic esters (U.S. Patent 5,385,909); gem-disubstituted esters
`(U.S. Patent 5,385,91 O); amino alkanoic esters
`(U.S. Patent 5,389,639);
`phosphorylcarbamate esters (U.S. Patent 5,391,730); carbamate esters (U.S. Patent
`5,411,967); carbamate esters (U.S. Patent 5,434,260); amidino carbamate esters
`(U.S. Patent 5,463,048); carbamate esters (U.S. Patent 5,480,988); carbamate esters
`(U.S. Patent 5,480,989); carbamate esters (U.S. Patent 5,489,680); hindered N-oxide
`esters (U.S. Patent 5,491,231 ); biotin esters (U.S. Patent 5,504,091 ); 0-alkyl ethers
`(U.S. Patent 5,665,772); and PEG esters of rapamycin (U.S. Patent 5,780,462). The
`preparation of these esters and ethers are disclosed in the patents listed above.
`
`Preferred 27-esters and ethers of rapamycin are disclosed in U.S. Patent
`5,256,790, which is hereby incorporated by reference. The preparation of these esters
`and ethers are disclosed in the patents listed above.
`
`Preferred oximes, hydrazones, and hydroxylamines of rapamycin are
`disclosed in U.S. Patents 5,373,014, 5,378,836, 5,023,264, and 5,563,145, which are
`hereby incorporated by reference. The preparation of these oximes, hydrazones, and
`hydroxylamines are disclosed in the above listed patents. The preparation of 42-
`oxorapamycin is disclosed in 5,023,263, which is hereby incorporated by reference.
`
`Particularly preferred rapamycins include rapamycin [U.S. Patent 3,929,992],
`CCl-779
`[rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic
`acid; U.S. Patent 5,362,718], and 42-0-(2-hydroxy)ethyl rapamycin [U.S. Patent
`5,665,772].
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`When applicable, pharmaceutically acceptable salts of the rapamycin can be
`formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric,
`tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric,
`hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic,
`benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable
`aids when the rapamycin contains a suitable basic moiety. Salts may also be formed
`from organic and inorganic bases, such as alkali metal salts (for example, sodium,
`lithium, or potassium) alkaline earth metal salts, ammonium salts, alkylammonium
`salts containing 1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon
`atoms in each alkyl group, and trialkylammonium salts containing 1-6 carbon atoms in
`each alkyl group, when the rapamycin contains a suitable acidic moiety.
`
`It is preferred that the mTOR inhibitor used in the antineoplastic combinations
`of this invention is a rapamycin, and more preferred that the mTOR inhibitor is
`rapamycin, CCl-779, or 42-0-(2-hydroxy)ethyl rapamycin.
`
`As described herein, CCl-779 was evaluated as a representative mTOR
`inhibitor in the mTOR inhibitor plus antimetabolite combinations of this invention.
`
`The preparation of CCl-779 is described in U.S. Patent 5,362,718, which is
`hereby incorporated by reference. When CCl-779 is used as an antineoplastic agent,
`it is projected that initial i.v. infusion dosages will be between about 0.1 and 100
`
`mg/m2 when administered on a daily dosage regimen (daily for 5 days, every 2-3
`
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`
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`
`weeks), and between about 0.1 and 1000 mg/m2 when administered on a once
`25 weekly dosage regimen. Oral or intravenous infusion are the preferred routes of
`administration, with intravenous being more preferred.
`
`30
`
`As used in accordance with this invention, the term "antineoplastic alkylating
`agent" means a substance which reacts with (or "alkyl ates") many electron-rich atoms
`in cells to form covalent bonds. The most important reactions with regard to their
`antitumor activities are reactions with DNA bases. Some alkylating agents are
`monofunctional and react with only one strand of DNA. Others are bifunctional and
`react with an atom on each of the two strands of DNA to produce a "cross-link" that
`covalently links the two strands of the DNA double helix. Unless repaired, this lesion
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`will prevent the cell from replicating effectively. The lethality of the monofunctional
`
`alkylating agents results from the recognition of the DNA lesion by the cell and the
`response of the cell to that lesion. (Colvin OM. Antitumor Alkylating Agents.
`In
`Cancer Principles & Practice of Oncology 5th Edition. ed. DeVita VT, Hellman S,
`
`5 Rosenberg SA. Lippincott Williams & Wilkins. Philadelphia 2001. p. 363.)
`Antineoplastic alkylating agents are roughly classified, according to their
`
`10
`
`15
`
`structure or reactive moiety, into several categories which include nitrogen mustards,
`such as mustargen, cyclophosphamide, ifosfamide, melphalan, and chlorambucil;
`
`azidines and epoxides, such as thiotepa, mitomycin C, dianhydrogalactitol, and
`dibromodulcitol; alkyl sulfonates, such as busulfan; nitrosoureas, such as
`bischloroethylnitrosourea (BCNU), cyclohexyl-chloroethylnitrosourea (CCNU), and
`
`methylcyclohexylchloroethylnitrosourea (MeCCNU); hydrazine and triazine derivatives,
`such as procarbazine, dacarbazine, and temozolomide; and platinum compounds.
`Platinum compounds are platinum containing agents that react preferentially at the N7
`position of guanine and adenine residues to form a variety of monofunctional and
`
`bifunctional adducts. (Johnson SW, Stevenson JP, O'Dwyer PJ. Cisplatin and Its
`
`Analogues. In Cancer Principles & Practice of Oncology 51h Edition. ed. DeVita VT,
`Hellman S, Rosenberg SA. Lippincott Williams & Wilkins. Philadelphia 2001. p.
`378.) These compounds include cisplatin, carboplatin, platinum IV compounds, and
`
`20 multinuclear platinum complexes.
`
`The following are representative examples of antineoplastic alkylating agents
`
`25
`
`of this invention.
`Meclorethamine is commercially available as an injectable (MUSTARGEN).
`Cyclophosphamide
`is
`commercially
`available
`as
`an
`injectable
`(cyclophosphamide,
`lyophilized CYTOXAN, or NEOSAR) and
`in oral
`tablets
`(cyclophosphamide or CYTOXAN).
`lfosfamide is commercially available as an injectable (I FEX).
`
`Melphalan is commercially available as an injectable (ALKERAN) and in oral
`tablets (ALKERAN).
`
`30
`
`Chlorambucil is commercially available in oral tablets (LEUKERAN).
`
`Thiotepa is commercially available as an injectable (thiotepa or THIOPLEX).
`Mitomycin
`is commercially available as an
`injectable
`(mitomycin or
`
`MUTAMYCIN).
`
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`Busulfan is commercially available as an injectable (BUSULFEX) and in oral
`tablets (MYLERAN).
`Lomustine (CCNU) is commercially available in oral capsules (CEENU).
`
`Carmustine (BCNU) is commercially available as an intracranial implant
`(GLIADEL) and as an injectable (BICNU).
`
`5
`
`Procarbazine is commercially available in oral capsules (MATULANE).
`Temozolomide is commercially available in oral capsules (TEMODAR).
`Cisplatin is commercially available as an injectable (cisplatin, PLATINOL, or
`PLATINOL'-AQ).
`Carboplatin is commercially available as an injectable (PARAPLATIN).
`
`10
`
`The following table briefly summarizes some of the recommended dosages for
`the antineoplastic alkylating agents listed above.
`
`15
`
`Table 1. Recommended Dosages of Antineoplastic Alkylating Agents
`
`Drug
`
`Mustargen
`
`Cyclophosphamide
`
`lfosfamide
`
`Melphalan
`
`Chlorambucil
`Thiotepa
`
`Mitomycin
`
`Busulfan
`
`Lomustine
`
`Dosage
`
`0.4 mg/kg
`
`40-50 mg/kg i.v.
`10-15 mg/kg i.v.
`3-5 mg/kg i.v.
`1-5 mg/kg oral
`
`1.2 g/m2 i.v.
`
`6 mg orally
`
`10 mg orally
`
`0.15 mg/kg orally
`
`16 mg/m2 i.v.
`
`0.1-0.2 mg/kg orally
`0.3-0.4 mg/kg i.v.
`
`Regimen
`
`each course given as a singe dose or in
`divided doses of 0.1 to 0.2 mg/kg/day.
`in divided doses over a period of 2-5 days
`every 7-1 0 days
`twice weekly
`daily
`
`daily for 5 consecutive days; repeated
`every 3 weeks or after recovery from
`hematologic toxicitv.
`daily for 2-3 weeks followed by 4 weeks
`rest, then 2 mg daily maintenance dosage
`daily for 7-10 days followed by 2 mg daily
`maintenance after white blood cell count
`has recovered.
`daily for 7 days, followed by a rest period
`of at least 14 days, then 0.005 mg/kg
`daily maintenance.
`single infusion over 15-20 minutes every
`2 weeks for 4 doses, followed by a rest
`then administered at 4 week
`period,
`intervals for maintenance.
`daily for 3-6 weeks
`every 1-4 weeks
`
`20 mg/m2 i.v.
`
`every 6-8 weeks
`
`1.8 mg/m2 orally
`
`daily
`
`130 mg/m2 orally
`
`every 6 weeks
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`Drug
`
`Carmustine
`
`Procarbazine
`
`Temozolomide
`
`Cisplatin
`
`Carboplatin
`
`Dosage
`
`Regimen
`
`150-200 mg/m2 i.v.
`
`every 6 weeks
`
`2-4 mg/kg orally
`
`1-2 mg/kg orally
`
`daily for first week, then 4-6 mg/kg until
`maximum response is achieved
`mainentance
`
`150 mg/m2 orally
`
`20 mg/m2 i.v.
`75-100 mo/m2 i.v.
`360 mg/m2 i.v.
`
`for 5 days per 28-day
`once daily
`treatment cycle
`daily for 5 days per cycle
`once every 4 week cycle
`once every 4 week cycle
`
`Preferred mTOR inhibitor plus antineoplastic alkylating agent combinations of
`this invention include CCl-779 plus cisplatin; CCl-779 plus cyclophosphamide; CCl-
`779 plus carboplatin; and CCl-779 plus BCNU.
`
`5
`
`The antineoplastic activity of the mTOR inhibitor plus antineoplastic alkylating
`agent combinations were confirmed using CCl-779 as a representative mTOR
`inhibitor in in vitro and in vivo standard pharmacological test procedures using
`combinations of CCl-779 plus cisplatin; CCl-779 plus cyclophosphamide; and CCl-779
`
`plus BCNU as representative combinations of this invention. The following briefly
`describes the procedures used and the results obtained.
`
`Human rhabdomyosarcoma lines Rh30 and Rh1 and the human glioblastoma
`line SJ-GBM2 were used for in vitro combination studies with CCl-779 and alkylating
`agents. In vivo studies used a human neuroblastoma (NB1643) and human colon line
`GC3.
`
`Dose response curves were determined for each of the drugs of interest. The
`
`cell lines Rh30, Rh1 and SJ-G2 were plated in six-well cluster plates at 6x103, 5x103
`and 2.5x1 o4 cells/well respectively. After a 24 hour incubation period, drugs were
`added in either 10%FBS+RPMI 1640 for Rh30 and Rh1 or 15%FBS+DME for SJ-G2.
`
`After seven days exposure to drug containing media, the nuclei were released by
`treating the cells with a hypotonic solution followed by a detergent. The nuclei were
`
`then counted with a Coulter Counter. The results of the experiments were graphed
`and the IC50 (drug concentration producing 50% inhibition of growth) for each drug
`was determined by extrapolation. Because the IC50s varied slightly from experiment
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`to experiment, two values that bracketed the IC50 of each drug were used in the
`interaction studies. The point of maximum interaction between two drugs occurs
`when they are present in a 1: 1 ratio if the isobole is of standard shape. Therefore,
`each of the three approximate IC50 concentrations of CCl-779 was mixed in a 1 : 1
`ratio with each of three approximated IC50s of cisplatin, BCNU, and melphanan. This
`resulted in nine 1 :1 combinations of drugs in each experiment plus three IC50
`concentrations for CCl-779 and the other drug. This protocol usually resulted in at
`least one combination for each drug containing an IC50 value. The 1 :1 combination of
`IC50 concentrations for CCl-779 and each chemotherapy drug was then used to
`calculate additivity, synergism, or antagonism using Berenbaum's
`formula:
`xJX50+yN 50,=1, <1,>1.
`If the three concentrations of CCl-779 tested alone didn't
`produce an IC that matched any of the three I Cs of the other compound tested alone,
`all the 1 :1 combinations were checked to see if their ICs fell between the appropriate
`I Cs of drugs tested singly. If they did, the effect was considered additive.
`The results obtained in the in vitro standard pharmacological test procedure
`showed when tested against Rh30 tumor line, the combination of CCl-779 plus
`cisplatin was synergistic; the combination was greater than additive but did not reach
`levels of being mathematically synergystic against the Rh1 tumor cell line, and was
`additive against the SJ-G2 tumor cell line. A combination of CCl-779 plus BCNU was
`synergistic against the SJ-G2 tumor cell line and greater than additive but did not
`reach levels of being mathematically synergystic against the Rh30 cell line, and
`additive against the Rh1 cell line. The combination of CCl-779 plus melphanan was
`additive against each of the cell lines.
`
`5
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`10
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`15
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`20
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`25
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`Female CBA/CaJ mice (Jackson Laboratories, Bar Harbor, ME), 4 weeks of
`age, were immune-deprived by thymectomy, followed 3 weeks later by whole-body
`
`irradiation (1200 cGy) using a 137Cs source. Mice received 3 x 106 nucleated bone
`
`30
`
`marrow cells within 6-8 h of irradiation. Tumor pieces of approximately 3 mm3 were
`implanted in the space of the dorsal lateral flanks of the mice to initiate tumor growth.
`Tumor-bearing mice were randomized into groups of seven prior to initiating therapy.
`Mice bearing tumors each received drug when tumors were approximately 0.20-1 cm
`in diameter. Tumor size was determined at 7-day intervals using digital Vernier
`calipers interfaced with a computer. Tumor volumes were calculated assuming tumors
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`5
`
`10
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`15
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`20
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`25
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`30
`
`to be spherical using the formula [(n/6) x d 3], where dis the mean diameter. CCl-
`
`779 was given on a schedule of 5 consecutive days for 2 weeks with this cycle
`repeated every 21 days for 3 cycles. This resulted in CCl-779 being given on days 1-
`5, 8-12 (cycle 1); 21-25, 28-32 (cycle 2); and 42-46, 49-53 (cycle 3). The schedule of
`the other chemotherapy drug for each study was as follows:
`
`Cyclophosphamide on days 1 and 8 every 21 days for 3 cycles
`
`The combination of CCl-779 and cyclophosphamide was evaluated using a
`human rhabdosarcoma (Rh18) using the mouse xenograft test procedure described
`In this test procedure, the effect of CCl-779 with cyclophosphamide (44
`above.
`mg/kg) was additive. When combined as suboptimum dosages, CCl-779 plus
`cyclophosphamide was equivalent to cyclophosphamide given at an optimum dosage.
`
`Based on the results of these standard pharmacological test procedures,
`combinations of an mTOR inhibitor plus an antineoplastic alkylating agent are useful
`as antineoplastic therapy. More particularly, these combinations are useful in the
`treatment of renal carcinoma, soft tissue sarcoma, breast cancer, neuroendocrine
`tumor of the lung, cervical cancer, uterine cancer, head and neck cancer, glioma, non-
`small cell lung cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma,
`small cell lung cancer, ovarian cancer, colon cancer, esophageal cancer, gastric
`cancer, leukemia, colorectal cancer, and unknown primary cancer. As these
`combinations contain at least two active antineoplastic agents, the use of such
`combinations also provides for the use of combinations of each of the agents in which
`one or both of the agents is used at subtherapeutically effective dosages, thereby
`lessening toxicity associated with the individual chemotherapeutic agent.
`
`In providing chemotherapy, multiple agents having different modalities of
`action are typically used as part of